Evolutionary Ways

by Merry Youle
 

…one would have to say that natural selection does not work as an engineer works. It works like a tinkerer – a tinkerer who does not know exactly what he is going to produce but uses whatever he finds around him…to produce some kind of workable object.
   – François Jacob, 1977 (1)
 

Evolution skeptics point to complex biological features as evidence that the amazing intricacy of the biological world could not be the product of blind mutation. The de novo  creation of such com­­plexity, they argue, would require far too many coordinated mutational changes before the new form provided any fitness benefit. Thus, evolution simply cannot explain such apparent leaps. Evo­lutionists, beginning with Darwin, and continuing today, have posited that complex features do not require breathtaking leaps, but rather arise through stepwise modification of existing forms. Such random tinkerings, each requiring comparatively few mutations, occasionally generate a new form that is slightly more complex. For this process to proceed further, the new form must benefit the organism. Only then can it be maintained in the population long enough to serve as a stepping-stone to yet greater complexity. The probability of success is low, and many of these experiments will lead to evolutionary dead-ends. Does evolution proceed in this haphazard way? Can small ran­dom steps add up to an evolutionary leap?
 

Taking a leap in silico

Evolution experiments in silico  say "yes." Here the evolving "organisms" are computer algorithms. Each digital organism possesses a heritable "genotype" that, when computationally expressed, yields a corresponding "phenotype." One such study by Lenski and colleagues (2) started with an ancestor with a circular genome of 50 "genes" that were expressed in sequence, generation after generation. Each gene was one of a set 26 executable instructions that included, among others, a copy instruction that carried out self-replication. No sex here, no horizontal gene transfer – only bi­nary fission and vertical inheritance. To simulate evolution, both heritable variation and selection were introduced. Variation arose from random mutation during genome replication. These muta­tions included substitutions in which one instruction was randomly replaced by another, as well as indels in which one instruction was inserted or deleted. Each genotype displayed a different phe­no­type with a particular replication efficiency and computational me­ta­bolism. Like biological me­ta­bolism, computational metabolism required energy.
 

Figure 1. Digital organism in Avida. Each in­di­vi­du­al orga­nism has a circular genome and a vir­tual CPU with two stacks and three registers that hold 32-bit strings. Execution of the ge­no­mic program generates a com­putational me­ta­bo­lism, whereby numerical substra­tes can be in­put from the environment, processed in stacks and registers, and resulting products output to the environment. Source

Some specific gene combinations, when expressed in the correct sequence, were able to perform one of nine logic functions of varying complexity. The ancestral genotype could perform none of the nine. Acquisition of even the simplest function by the ancestor required more than one mutation. The more complex the function, the more mu­tational steps required for even the most direct evo­lu­ti­o­na­ry route. Each organism was rewarded with energy packets for each function it performed during its "life­time." The more complex the function, the more packets were awarded. Increased genome length also earned energy packets. These energy boosts, in turn, increased the organism's reproductive fitness. Over the course of hundreds or thousands of generations, driven by "se­lec­tion" based on "reproductive fitness," a dominant or­gan­ism evolved that was capable of performing all nine logic functions, including the most complex.
 

To explore what this in silico  work can tell us about the evolution of complex features, the researchers focused on the acquisition of the most complex of the nine logic functions. Theoretically, the ancestor could gain this capability through a minimum of 16 mutations. As you would expect, the random paths taken by this simulated evolution were less direct; likewise, success was not guaranteed. However, that complex capability evolved in 23 out of 50 identical experiments. Among those 23, the most direct route comprised 51 steps; the longest one, 721 steps – a step being each time the offspring differed from the parent. Some of those steps were single mutations, some double or triple. The route was not a steady upward trajectory toward greater fitness; some steps were lateral, some were even deleterious relative to the parent. Conclusions? The probability of evolution of a bene­fi­cial complex logic function was high even when the route spanned several hundred small steps. Although this achievement was repeatable, it is extremely unlikely that it would ever arise by the same convoluted path twice.
 

An additional series of similar experiments tested the hypothesis that the intermediate steps en route to evolution of a complex function must themselves confer a fitness benefit. The research­ers ran 50 experiments under "environmental conditions" that did not energetically reward any of the eight simpler functions. Energy packets would be given only for carrying out the most complex logic function. The result ? Zero out of 50 achieved that capability.
 

This in silico  work provides supportive evidence that more complex features can evolve given the prior evolution of simpler intermediate features that are, themselves, beneficial. Nevertheless, it is quite a leap from in silico  to in vivo. It would surely be more convincing to a skeptic if we could point to an in vivo  example of a similar leap. Time to call on the phage !
 

A successful leap in vivo

Figure 2. Electron micrograph of phage UrLamb­da by Ro­bert Duda. Unlike today's common lab strain, its pro­bable ancestor has six side tail fi­bers that participate, along with the tail tip, in host recognition and ad­sorp­tion. Source

Phage λ has been studied intensively for decades and decades, more than any other phage. The particular do­mesticated strain propagated in most labs lacks the side tail fibers of the ancestral wild-type phage, fibers that functioned in host recognition. As a result, this λ strain is absolutely dependent on the receptor binding protein (RBP) at the tip of its tail for adsorption to its E. coli  host. That RBP recognizes and binds to a specific porin, LamB, located in the cell's outer membrane. Subsequently λ's chromosome crosses that first membrane through this porin. Because LamB is part of E. coli's maltose transport system, this frugal bacterium synthesizes it only when it needs to import maltose for its carbon and energy source. When E. coli  is grown in the lab on glucose, LamB syn­the­sis is repressed and consequently λ's adsorption rate is barely detectable. Throughout its long lab history, λ has never been known to adsorb to another receptor. Could it possibly do that? To ans­wer this, researchers gave λ a strong incentive to take that leap, and then closely observed what happened.
 

The incentive was indeed strong, and prolonged. Meyer and colleagues (3) cultured λ together with E. coli  for 28 days in a medium that contained glucose, but no maltose. Every day they transferred 1% of the culture into fresh medium. E. coli  grows well under such conditions, but λ barely persists. A small phage population (1 virion per 10² to 10³ cells) subsists by infecting the small number of bacteria that spontaneously synthesize LamB. This placed the phage under very strong selection pressure to evolve a workaround. This λ did – by quickly and repeatedly evolving the ability to use a different receptor. Quickly here means within 12 days on average under these experimental con­ditions. Repeatability was demonstrated by λ's success in 24 out of 96 replicate experiments. If this was so easy for the phage, you might be wondering why no one had observed this previously. The answer: anyone testing λ's ability to use other receptors would have ended their experiments after hours, not days or weeks. In other words, researchers had asked what were the receptor-bind­ing capabilities of their phage stock, not what abilities could the phage evolve under extreme duress.
 

Did all 24 winners find the same solution? All did evolve to use the same receptor, porin OmpF. To a researcher predicting λ's new receptor choice, OmpF would have seemed the most likely can­di­da­te because this porin is the one most similar structurally to LamB. Moreover, unlike LamB and most porins, OmpF is synthesized constitutively, thus always available to the phage. Genome se­quencing demonstrated that all 24 winners had also evolved essentially the same mechanism; they all possessed a set of four mutations located within the same four narrow regions within the RBP gene.
 

Granted, this change of receptor was a small leap that involved only one phage protein, but it was still a leap. All four mutations were required before the phage was able to use OmpF. This suggests that all four must have occurred simultaneously. However, the probability of this happening within 12 days in these small phage populations was so low as to defy even phage ingenuity. Further­mo­re, this was not the achievement of one exceptional phage, a singular event not to be repeated. Rather it was achieved by 24 out of 96 replicate cultures. Something else must have been going on to account for this rapid and consistent success.
 

(Click to enlarge )
Figure 3. Steps in the co­evo­lu­tion of phage λ and its E. coli host leading to the phage’s ability to target a new receptor, OmpF. 1) The ancestral phage targets the LamB porin using the J protein (receptor binding protein) and injects its DNA into the periplasm, then the DNA is transported into the cytoplasm via the ManXYZ permease. 2) The bacteria evolve re­sist­ance by muta­tions in malT, a positive regulator of LamB expression. 3) However, spontaneous inductions of LamB generate a subpopulation of phenotypically sensitive cells that can sustain the phage population. 4) The phage evolves mu­ta­tions in the J protein that improve per­for­man­ce on the LamB receptor. Some of these mu­ta­tions are also required for the phage to target OmpF. 5) The phage eventually evolves the four mu­ta­tions that enable it to use OmpF. 6) How­ev­er, the bacteria may evolve ad­dition­al re­sist­ance by mutations in manY or manZ that pre­vent transport of the phage DNA into the cyto­plasm. When these mutants become suf­fi­cient­ly common, there is little or no benefit to mu­tant phage that can use OmpF. Source

To explore this puzzle, the researchers took a closer look at the ongoing interactions between phage and host over the course of these experiments. Faced with long-term maltose-free conditions, the phage population was de­spe­ra­te to exploit whatever LamB was made by the bac­teria. The host, having no need for maltose but being sub­ject­ed to persistent phage attack, evolved tighter re­pression of LamB synthesis in all of the cultures. This further re­duced the minimal amount of LamB syn­the­si­zed, and thereby decreased the number of receptors exposed on the cell surface. Now a phage colliding with a bacterium was less likely to locate a receptor. This, in turn, increas­ed the pressure on the phage to convert as many of those lucky hits as possible into successful binding and chromosome delivery.
 

The researchers found that the phage achieved improved adsorption to LamB by mutations in the RBP gene before they had acquired the ability to use OmpF. The evidence ? All of these RBP mutations were nonsynonymous chan­ges, which indicated that they were under positive se­lect­ion. They all clustered at specific points in the RBP struc­ture that interacted closely with LamB. Lastly, very similar mutations were found repeatedly among the successful phage lineages. Therefore, the researchers concluded that λ had not made one giant leap to OmpF binding, but rather had progressed by a series of mutational steps that themselves con­ferred immediate benefit while the phage was still dependent on LamB as its receptor.
 

Figure 4. Stepping stones across Dick Brook in Shrawley Wood. Credit: Philip Halling. Source

This begs another question. If it was this easy, with bene­ficial stepping-stones all along the way, why did only one in four phage lineages succeed ? One possible reason con­sidered was that some of the mutations that yielded an immediate benefit also hindered evolution of OmpF-bind­ing ca­pa­bility. No evidence was found for this in further experiments. Instead, the researchers found that the pha­ge switch to OmpF had been blocked by host mutations that arose during the ex­pe­ri­ment. These included muta­tions that altered two other proteins in the E. coli   malto­se transport system, ManY and ManZ. These proteins form maltose transport channels through the cell mem­brane, channels λ also uses for passage of its chromosome from the periplasm into the cytoplasm. Thus, these host muta­tions obstructed λ infection no matter which receptor was used; they even drove the phage to extinction in some cul­tures. Thus, whether λ made the leap to OmpF or failed was dependent not only on the phage, but also on the dy­namic co-evolution of its host.
 

Is there a take-home message here ? Both of these expe­ri­mental approaches support the hypothesis that evo­lu­tionary leaps are, in actuality, a sequence of small, beneficial steps – but caution ! The inviting stepping-stones-across-a-burbling-brook analogy is misleading. Imagine stepping blindly is every direction; imagine stepping-stones that continually shift or disappear entirely. Even the de­sti­na­tion shore doesn't stay put. Environmental conditions fluctuate. Co-evolution is forever moving the goalposts. The possibilities are endless. The outcome of any step is uncertain, but here we are, and the evolutionary dance continues.
 

References

(1) Jacob F. 1977. Evolution and tinkering. Science, 196 (4295), 1161 – 1166. PMID 860134 (Paywall; Open Access PDF here)

(2) Lenski RE, Ofria C, Pennock RT, and Adami C. 2003. The evolutionary origin of complex features. Nature, 423 (6936), 139 – 144. PMID 12736677 (Paywall; Open Access PDF here)

(3) Meyer JR, Dobias DT, Weitz JS, Barrick JE, Quick RT, and Lenski RE. 2012. Repeatability and con­tingency in the evolution of a key innovation in phage lambda. Science, 335 (6067), 428 – 432. PMID 22282803 (Paywall; Open Access PDF here)

 
 

 
 

 
 
 
Merry Youle is a phageophilic microbiology writer/editor and STC blogger emerita living on the Big Island of Hawaii. Her books in­clude the recently-published  Thinking Like a Phage and the 2015 title, Life in Our Phage World. This post is excerpted from her book in progress, Phage in Community.